Challenges in Predictive Modeling of Single Pass Tangential Flow Filtration for Continuous Biomanufacturing

Opportunities for process intensification and increased productivity have made the field of Continuous Biomanufacturing an area of high interest and active research. Within the purification train of producing biologics, Tangential Flow Filtration (TFF) is typically employed after chromatographic separations, to increase drug substance concentration, making the process more economical and further meeting dosage specifications. In a batch operation, concentration occurs via recirculation of the feed material where desired output concentration is attained through multiple pump-passes over the TFF membrane, while steadily excluding the buffer. Single-Pass Tangential Flow Filtration (SPTFF) enables continuity of this process by achieving similar concentration factors through a single – pass over these membranes while operating at low feed flow rates. Our work elucidates the development of a mechanistic process model to predict SPTFF performance across a relatively wide design space using a first principles approach. The developed model is found to be accurate for a range of high feed flow rates but is inaccurate at flow rates below 25 L/m2/hr. At very low flow rates, small differences in the mass transfer coefficient have been observed to significantly alter the prediction of the retentate concentration. We thus describe the challenges in predictive process modeling of SPTFF in antibody biomanufacturing.

Liquid–Solid Flow Characteristics in Vertical Swirling Hydraulic Transportation with Tangential Jet Inlet

In order to improve the efficiency and safety of vertical hydraulic transport systems for non-spherical particles, a new pipeline transport system with a tangential jet inlet is adopted in this study, and a modified non-spherical drag coefficient model is used to analyze the liquid–solid flow characteristics based on the CFD-DEM (Computational Fluid Dynamics-Discrete Element Method)coupling method. The focus of the study is on the influence of different tangential flow proportions in terms of the velocity distribution, the vorticity, the total pressure, the concentration and drag force of particles of various shapes. The conveying efficiency is measured according to the fluid velocity distribution and the particle concentration, and the safety of conveying is evaluated according to the flow structure and drag force of the particles. The result shows that the velocity of the swirling pipes is significantly higher than the straight pipe. With the increase of the tangential flow proportion, the swirling number and the vorticity magnitude increase, and the vortex core is broken and merged more quickly. Furthermore, the concentration gap and axial drag force gap between particles of various shapes are reduced with the effect of swirling flow, the particle concentration increases, and the particles of each component are uniformly mixed and transported.

Cell culture–based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act “universally” against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key points• An automated perfusion process for production of IAV DIPs was established.• DIP titers of 7.40E + 9 plaque forming units per mL were reached.• A novel tubular cell retention device enabled continuous virus harvesting.

Cytoplasmic tail truncation of SARS-CoV-2 Spike protein enhances titer of pseudotyped vectors but masks the effect of the D614G mutation

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV) and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein’s function. Here, we optimized concentration methods for SARS-CoV-2 Spike pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers, but had no impact on sensitivity to convalescent serum inhibition. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but that this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher titer vectors are required.